Use of deprenyl to rescue damaged nerve cells

ABSTRACT

The present invention relates to the use of deprenyl or derivatives or analogues of deprenyl to rescue damaged nerve cells in an animal; to pharmaceutical compositions containing deprenyl adapted for such use; and, to methods for the treatment of disorders of the nervous system by rescuing damaged nerve cells.

This application is a continuation of U.S. Ser. No. 07/929,579 filedAug. 14, 1992abandoned, which is a continuation-in-part of U.S. Ser. No.07/772,919 filed Oct. 8, 1991 abandoned, which is a continuation-in-partof U.S. Ser. No. 07/751,186 filed Aug. 26, 1991 abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/678,873 filed Apr. 4, 1991.

FIELD OF THE INVENTION

The present invention relates to the use of deprenyl or derivatives oranalogues of deprenyl to rescue damaged nerve cells in an animal; topharmaceutical compositions containing deprenyl adapted for such use;and, to methods for the treatment of disorders of the nervous system byrescuing damaged nerve cells in an animal. The invention also relates tomethods for testing drugs for their activity in rescuing nerve cells inan animal.

BACKGROUND OF THE INVENTION

Deprenyl (also referred to herein as selegiline orR-((-)-N,α-Dimethyl-N-2-propynyl phenethylamine) was first used as anadjunct to conventional drug therapy (L-dihydroxyphenylalanine (L-DOPA)plus a peripheral decarboxylase inhibitor) of Parkinson's disease (PD)in Europe over a decade ago on the basis that as a selective monoamineoxidase-B (MAO-B) inhibitor, it would elevate brain dopamine levels andpotentiate the pharmacologic action of dopamine formed from L-DOPA, andyet prevent the tyramine-pressor effect observed with non-selective MAOinhibitors. The combined drug therapy was reported to prolong theanti-akinetic effects of L-DOPA, resulting in the disappearance ofon-off effects, reduced functional disability, and increasedlife-expectancy in PD patients (Bernheimer, H., et al., J. Neurolog.Sci., 1973.20: p. 415-455,.Birkmayer, W., et al., J. Neural Transm.,1975. 36: p. 303-336, Birkmayer, W., et al., Mod. Prob.pharmacopsychiatr., 1983. 19: p. 170-177, Birkmayer, W. and P. Riederer,Hassler, R. G. and J. F. Christ (Ed.) Advances In Neurology, 1984.40(Y): p.0-89004, and Birkmayer, W., et al., J. Neural Transm., 1985.64(2): p. 113-128).

Studies examining deprenyl as an adjunct to conventional L-DOPA therapyhave reported a short term benefit which was usually lost by 1 year orless. Some, but not all, have reported that the levodopa dose can bedecreased when taken in conjunction with deprenyl (Elizan, T. S., etal., Arch Neurol, 1989. 46(12): p. 1280-1283, Fischer, P. A. and H.Baas, J. Neural Transm. (suppl.), 1987. 25: p. 137-147, Golbe, L.I.,Neurology, 1989. 39:p. 1109-1111, Lieberman, A. N. et al., N.Y. State J.Med., 1987. 87: p. 646-649, Poewe, W., F. Gerstenbrand, and G.Ransomayr, J. Neural Transm. (suppl.), 1987. 25: p. 137-147, Cedarbaum,J. M., M. Hoey, and F. H. McDowell, J. Neurol Neurosurg Psychiatry,1989. 52(2): p. 207-212, and Golbe, L. I., J. W. Langston, and I.Shoulson, Drugs, 1990.39(5): p. 646-651).

Increasingly deprenyl is being administered to Parkinson's diseasepatients following reports (parkinson, S. G. Arch Neurol 46, 1052-1060(1989) and U.S.A., P.S.G. N. Engl. J. Med. 321, 1364-1371 (1989)) thatit delays the disease's progression; however, no satisfactory mechanismhas been proposed to explain its action.

Support for the use of deprenyl in Parkinson's disease (PD) is largelybased on the findings of the DATATOP project (Parkinson, S. G. ArchNeurol 46, 1052-1060 (1989) and U.S.A., P.S.G.N. Engl. J. Med. 321,1364-1371 (1989)). This multicentre study reported that deprenyl delaysthe onset of disabling symptoms requiring additional pharmacotherapy bynearly one year; these. findings were reproduced in an independent butsmaller study (Tetrud, J. W. & Langston, J. W. Science 245, 519-522(1989)). Unfortunately, the design of the DATATOP study and itsconclusions have come under strong criticism (Landau, W.M. Neurology 40,1337-1339 (1990). Furthermore, while the authors of these projects statethat their results are consistent with the hypothesis that deprenylslows the progression of PD (Parkinson, S. G. Arch Neurol 46, 1052-1060(1989), U.S.A., P.S.G. N. Engl. J. Med. 321, 1364-1371 (1989) andTetrud, J. W. & Langston, J. W. Science 249, 303-304 (1990)), "they byno means constitute proof" (Tetrud, J. W. & Langston, J. W. Science 249,303-304 (1990)).

It has been proposed that deprenyl, an MAO-B inhibitor, may delay theprogression of PD by minimizing free-radical induced death of survivingdopaminergic nigrostriatal (DNS) neurons (Langston, J. W. in Parkinson'sDisease and Movement Disorders (eds. Jankovic, J. & Tolosa, E.) 75-85(Urban and Schwarzenberg, Baltimore-Munich 1988)) based on theobservation that deprenyl could block MPTP-induced neurotoxicity inprimates (Langston, J. W., Forno, L. S. Robert, C. S. & Irwin, I. BrainRes 292, 390-394 (1984)) and the hypothesis that other environmentaltoxins with mechanisms of action similar to that of MPTP may be involvedin the etiology of PD (Tanner, C. M. TINS 12, 49-54 (1989)). However,since MAO-B is not present in dopaminergic neurons (Vincent, S. R.Neuroscience 28, 189-199 (1989), Pintari, J. E., et al. Brain Res 276,127-140 (1983), Westlund, K. N., Denney, R. M., Kochersperger, L. M.,Rose, R. M. & Abell, C. W. Science (Wash D.C.) 230, 181-183 (1985) andWestlund, K. N., Denney, R. M., Rose, R. M. & Abell, C. W. Neuroscience25, 439-456 (1988)), it is unclear how its inhibition would protect DNSneurons unless another highly toxic compound were formed innon-dopaminergic neurons which could in turn damage DNS neurons in amanner analogous to that of MPTP. Surprisingly, no investigation hasincluded the measurement of DNS neuronal numbers to determine whetherdeprenyl could influence neuronal survival when measured after MPTP hasbeen cleared from the central nervous system.

SUMMARY OF THE INVENTION

Broadly stated the present invention relates to the use of deprenyl or aderivative of deprenyl, or an analogue of deprenyl to rescue damagednerve cells in a patient.

The invention also relates to a pharmaceutical composition for use inthe treatment of disorders of the nervous system comprising an amount ofdeprenyl, a derivative of deprenyl, or an analogue of deprenyl,effective to rescue damaged nerve cells in a patient.

The invention further relates to a method for the treatment of disordersof the nervous system by rescuing damaged nerve cells in a patientcomprising administering to a patient an amount of deprenyl, aderivative of deprenyl, or an analogue of deprenyl, effective to rescuedamaged nerve cells.

The invention also relates to methods for testing a drug for activity inrescuing damaged nerve cells.

The terms "rescue of damaged nerve cells" or "rescuing of damaged nervecells" herein refers to the reversal of the sequence of damage to deathin lethally damaged nerve cells and/or compensation in part for the lossof muscle-derived trophic support.

In accordance with one embodiment of the invention a method for testinga drug for activity in rescuing nerve cells comprising administering anagent having neurotoxic activity to a test animal; administering thedrug to the test animal; sacrificing the test animal within a period of20 days from completion of administration of the agent; taking serialsections of the brain through the substantia nigra compacta; determiningthe number of tyrosine hydroxlyase positive somata in alternate serialsections of the brain through the substantia nigra compacta anddetermining the number of Nissl stained positive substantia nigracompacta in intervening serial sections; and comparing the number oftyrosine hydroxlyase positive somata and Nissl stained somata determinedwith the number of tyrosine hydroxlyase positive somata and Nisslstained somata in serial sections of the brain through the substantianigra compacta of a control animal which has not been administered thedrug.

In accordance with another embodiment of the invention a method fortesting a drug for activity in rescuing damaged nerve cells in a patientis provided comprising carrying out an axotomy on a test animal;administering the drug to the test animal; determining the number ofcholine acetyl transferase positive somata for histological sectionsfrom the site of the axotomy; and comparing the number of choline acetyltransferase positive somata determined with a number of choline acetyltransferase positive somata in serial sections from the site of theaxotomy in a control animal which has not been administered the drug.

BRIEF DESCRIPTION OF THE DRAWINGS

Further details of the invention are described below with the help ofthe examples illustrated in the accompanying drawings in which:

FIG. 1 shows a comparison of the known molecular structures ofL-deprenyl, clorgyline and pargyline;

FIG. 2 is a graph showing the numbers of tyrosine hydroxylaseimmunopositive (TH+) neurons in the substantia nigra compacta (SNc)following the administration of MPTP.;

FIG. 3 are joint plots of the counts of TH+ and Nissl stained SNc somatafrom corresponding areas of immediately adjacent sections for SalineOnly treated (A1,A2,A3), MPTP-Saline treated (B1,B2,B3) andMPTP-Deprenyl treated animals (C1,C2,C3) with the data pooled from 3animals in each group at 20 days following the MPTP treatment;

FIG. 4 are joint Nissl/TH+ plots for days 0, 3, 5, 10, 15 and 20 forpooled saline controls;

FIG. 5 are joint Nissl/TH+ plots for cumulated saline controls and fordays. 0,5,10,15, and 20 after completion of MPTP treatment;

FIG. 6 shows superimposed plots for the percentage of Nissl stainedsomata and the percentage of TH+ immunoreactive somata relative to themean values for the saline controls for Day 0 through Day 60;

FIG. 7 is a graph showing the cumulative counts of TH+ SNC neuronsversus section number for individual representative SNc nuclei takenfrom alternate 0 micron serial sections throughout the entire nucleus;

FIG. 8 is a graph showing the mean and SEM values for the MPTP,MPTP-Saline and MPTP-deprenyl treated mice;

FIG. 9 is a graph showing TH+ somal counts for SNC neurons along therostrocaudal length of a nucleus;

FIG. 10 is a graph showing the mean corrected number of TH+ somata forsaline, MPTP, MPTP-saline, MPTP-clorgyline and MPTP-deprenyl treatedanimals with a table illustrating the timing of the various treatments;

FIG. 11 is a bar graph showing MAO-A and MAO-B measurements at 24 hours(d4) after the first administration of deprenyl (0.25 mg/kg or 0.01mg/kg) and 8 days later (d22);

FIG. 12 shows a spectral analysis of locomotory activity for miceinjected with MPTP;

FIG. 13 shows high resolution power spectra for LD and DD preinjectioncontrol period from a saline injected mouse;

FIG. 14 shows a high resolution power spectra for control and MPTP mice;

FIG. 15 is a graph showing the normalized sum peak power versus medianday;

FIG. 16A and 16B show SNc sections for glued brains from animals treatedwith MPTP or saline;

FIG. 17A, B, C, and D are graphs showing the counts of TH+ SNc and VTAneuronal somata following MPTP treatment taken through whole nucleiexpressed as a percentage of the mean counts for the correspondingsaline-injected animals (A); the concentration of striatal DA (B); theconcentration of striatal DOPAC, and the DOPAC/DA ratio (D) for salineand MPTP injected mice; and

FIG. 18 is a graph showing the mean OD/mean O.D. for saline backgroundversus days after MPTP injections;

FIG. 19 shows photomicrographs of adjacent ChAT immunoreacted (A1 andB1) and Nissl stained (A2 and B2) sections through the facial nucleusipsilateral to transection of the facial nerve;

FIG. 20 is a bar graph for the counts of ChAT+ somata for the facialnuclei for the different lesion and treatment groups (bars-means, errorbars-standard deviations);

FIG. 21 are graphs showing Joint Nissl/ChAT+ counts of adjacent sectionsfor the no lesion groups (FIG. 14A), the ipsilateral lesion-salineanimals (FIG. 14B), the lesion-deprenyl animals (FIG. 14B), and thecontralateral lesion animals (FIG. 14C);

FIG. 22 shows ChAT+ counts for facial motoneurons in 35 day old ratsafter a unilateral axotomy at 14 days of age;

FIG. 23 shows the data shown in FIG. 20 and includes data for additionalanimals;

FIG. 24 shows the counts of TH+ SNc somata following treatment withdeprenyl;

FIG. 25 shows the data shown in FIG. 23 and includes data from animalstreated with N-(2-aminoethyl)-4-chlorobenzamide.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have studied the time course of neuronal deathinduced by the neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine(MPTP). MPTP is oxidized, under the action of monoamine oxidase-B(MAO-B), via a dihydropyridium intermediate (MPDP+) to its toxicmetabolite 1-methyl-4-phenyl-pyridinium ion (MPP+). It is believed thatMPTP is converted to MPP+ in nondopaminergic cells, released and thentaken up into dopaminergic neurons where it exerts its neurotoxiceffects (see Vincent S. R. Neuroscience, 1989, 28 p. 189-199, Pintari,J. E. et al. Brain Res, 1983, 276(1) p. 127-140, Westlund, K. N. et al.Neuroscience, 1988, 25(2) p. 439-456, Javitch, J. A. et al. P.N.A.S. USA1985, 82(7) P. 2173-2177, Mayor, 1986 #1763, and Sonsalia, P. K. et al.17th Annual Meeting Of The Society For Neuroscience, New Orleans, LA.,USA, November, 1987, 13(2)).

MPTP is rapidly metabolized and cleared in the mouse (Johannessen, J. N.et al. Life Sci. 1985, 36: p. 219-224, Markey, S. P. et al. Nature,1984, 311 p. 465-467, Lau, Y. S. et al. Life Sci. 1988, 43(18): p.1459-1464). In contrast to the rapid metabolism and excretion of MPTP,the present inventors have demonstrated that loss of dopaminergicneurons progresses over a period of twenty days following cessation ofMPTP administration. MPTP (30 mg/kg/d) was administered i.p. to mice forfive consecutive days (total cumulative dose 150 mg/kg) to produce aloss of approximately 50% of TH-immunopositive (TH+) neurons in thesubstantia nigra compacta (SNc) and ventral tegmental area (VTA)(seeSeniuk, N. A., W. G. Tatton, and C. E. Greenwood, Brain Res., 1990. 527:p. 7-20 which are incorporated herein by reference for the relationshipbetween MPTP dose and loss of catecholaminergic neurons). The presentinventors have also found that the death of TH+ Snc neurons followed asimilar timecourse 20-30% of TH+ somata were lost by the five days afterthe completion of the administration of MPTP; loss of TH+ neuronscontinued over the next ten to fifteen days with no detectable lossthereafter. This continual loss of TH+ neurons could not be accountedfor by the presence of MPP+, based on the excretion data referred toabove. Joint plots of counts of TH+ and Nissl stained SNc somata alsoconfirmed that the loss of TH+ somata represented the death of SNcneurons rather than a loss of TH immunoreactivity.

In tandem with the loss of TH+ SNc somata the present inventors havealso found changes in immunodensity of TH protein in SNc and the ventraltegmental area (VTA). Cytoplasmic TH immunodensity was 40% lower in thesomata of the remaining TH+ DNS neurons for MPTP-treated animals at day5 in comparison to saline treated controls. Average somalTH-immunodensity increased over time and had reached control levels by20 days following MPTP. Alterations in striatal DA concentrations anddopamine-dependent behaviours such as locomotion were found to parallelthe changes in TH-immunochemistry. Further, the present inventors foundthat an increase in striatal DA content and DA synthesis as estimated byDOPAC/DA ratios also appeared to parallel behavioural recovery andindicated increased DA content and synthesis in the VTA and SNc neuronssurviving MPTP exposure.

Thus, the present inventors have significantly found that followingMPTP-induced neuronal damage, there is a critical 20 day period in whichTH+ SNc neurons either undergo effective repair and recovery or elsethey die.

Most studies with deprenyl have been designed to demonstrate thatinhibition of MAO-B activity in vivo blocks the conversion of MPTP toMPP+ and the neurotoxicity of MPTP. As a consequence, deprenyl wasusually given either several hours or for several days prior to and thenthroughout MPTP administration to ensure that MAO-B activity wasinhibited during the time of MPTP exposure (for example, see Cohen, G.,et al., Eur. J. Pharmacol., 1984. 106: p. 209-210, Heikkila, R. E., etal., Eur. J. Pharmacol, 1985. 116(3): p. 313-318, Heikkila, R. E., etal., Nature, 1984. 311: p. 467-469 and Langston, J. W., et al., Science(Wash. D.C.), 1984. 225 (4669): p. 1480-1482). Comparable results havebeen obtained using other selective inhibitors of MAO-B such as AGN1133,AGN-1135 and MD 240928 (Heikkila, R. E., et al., Eur. J. Pharmacol,1985. 116(3): p. 313-318 and Fuller, R. W. and L. S. K. Hemrick, LifeSci, 1985.37(12): p. 1089-1096) suggesting that the mechanism of actionof deprenyl was mediated by its ability to block MAO-B and therebyprevent the toxin from being converted to its active form.

In contrast to the above studies, the present inventors were interestedin determining whether deprenyl could exert an effect on DSN neuronswhich was independent of its ability to block MPTP conversion to MPP+.MPTP-treated mice (cumulative dose of 150 mg/kg) received deprenyl(0.01; 0.25, 10 mg/kg i.p.; 3 times per week) from day 3 to day 20following MPTP administration. Deprenyl administration was withhelduntil day 3 to ensure that all mice were exposed to comparable levels ofMPP+ and that all MPTP and its metabolytes had been eliminated from thecentral nervous system. Clorgiline, an MAO-B inhibitor, was alsoadministered to the MPTP-treated mice.

The present inventors found that in saline treated mice, about 38% ofdopaminergic substantia nigracompacta (DSN) neurons died progressivelyover the twenty days. The number of DSN neurons was found to bestatistically the same in the MPTP-Saline and MPTP-Clorgiline treatedmice. However, deprenyl increased the number of DSN neurons survivingMPTP-induced damage (16% loss--0.01 mg/kg, 16% loss--0.25 mg/kg, and 14%loss 10mg/kg), with all doses being equipotent. Thus, the presentinventors have demonstrated that deprenyl could rescue dying neurons andincrease their probability of undergoing effective repair andre-establishing their synthesis of enzymes, such as tyrosinehydroxylase, necessary for dopamine synthesis. This is believed to bethe first report of a peripherally or orally administered treatmentwhich reverses the sequence of damage to death in neurons which wouldhave otherwise died.

The inventor's studies ruled out the possibility that deprenyl wasmediating its resuscitative effect through inhibition of MPTP conversionto its toxic metabolite MPP+. The results suggest that deprenyl has apreviously unidentified mechanism of action. It is difficult toreconcile a direct effect of deprenyl in dopaminergic neurons themselvesdue to the absence of MAO-B in these cells (Vincent, S. R., Neuroscience28, 189-199 (1989); Pintari, J. E., et al. Brain Res. 276, 127-140(1983); Westlund, K. N. et al. Science, (Wash D.C.) 230, 181-183 (1988)and Westlund, K. N. et al. Neuroscience 25, 439-456 (1988)), making itunlikely that the results can be explained on the basis of MAO-Binhibition by deprenyl within the dopaminergic neurons themselves.Measurements of MAO-A and MAO-B in MPTP mice at the beginning and end oftreatment with deprenyl (0.01 mg/Kg) showed that the 0.01 mg/Kg dose didnot produce any significant MAO-A or MAO-B inhibition at the two timeperiods, suggesting that it is highly unlikely that deprenyl mediatesits resuscitative effect thought inhibition of MAO-B. Further,clorgyline an MAO-A inhibitor did not increase the number of survivingDSN neurons after neuronal death induced by MPTP.

Other results have confirmed that the rescue of damaged neurons bydeprenyl does not depend on the known MAO-B or MAO-B inhibitionactivity. It has been demonstrated that the rescue of axotomizedmotoneurons by deprenyl (see discussion below) is permanent as themotoneurons do not die when the deprenyl treatment is subsequentlydiscontinued. It has also been demonstrated that the MAO-B inhibitorN-(2-aminoethyl)-4-chlorobenzamide hydrochloride is not effective inrescuing damaged motoneurons.

The survival of rat facial motoneurons after axotomy at 14 days of agewas also examined and it was found that deprenyl increased by 2.2 timesthe number of motoneurons surviving 21 days after the axotomy (SeeExample 3 herein). Further, a dose of 0.01 mg/kg of deprenyl was Just aseffective as 10 mg/kg deprenyl in rescuing the motoneurons similar tothe 0.01 mg/kg dose used with the MPTP model. Pargyline has also beenshown to rescue motoneurons. Thus, it has been significantlydemonstrated that deprenyl and pargyline can compensate in part for theloss of trophic support caused by axotomy suggesting a role for deprenyland its analogues and derivatives in the treatment of motoneuron deathin conditions such as amyotrophic lateral sclerosis.

Animals lesioned at 14 days, treated for the next 21 days with 10 mg/kgdeprenyl (d14-35) and then left untreated until 65 days of age did notshow any further motoneurons death. It was also demonstrated, that therescue is permanent for the axotomized motoneuron i.e. the motoneuronsdo not begin to die when deprenyl treatment is discontinued after 21days and there is no further death over the next 30 days.

The resuscitative effect of deprenyl may be mediated by any of the cellsin the nervous system and the mechanism likely involves the activationof a receptor on the cells (such as a receptor for a neuronotrophicfactor) through a structure which may not be related to the structurewhich blocks MAO-B. This would imply that deprenyl could help preventthe death of all neurons in the brain that respond to glial trophicfactors, rather than just influencing dopaminergic neurons alone. Henceas well as being therapeutically effective in Parkinson's disease, itwould also be effective in other neurodegenerative and neuromusculardiseases and in brain damage due to hypoxia, ischemia, stroke or traumaand may even slow the progressive loss of neurons associated with brainaging (Coleman, P. D. & Flood D. G., Neurobiol. Aging 8, 521-845 (1987);McGeer, P. L. et al. in Parkinsonism and Aging (eds. D. B. Calne, D,C, -G. Comi and R. Horowski). 25-34 (Plenum, New York, 1989). It may also beuseful in stimulating muscle reinnervation in traumatic and nontraumaticperipheral nerve damage.

The present studies also indicate that the propargyl terminus may be afactor required for the rescue of damaged neurons. As indicated above,the MAO-A inhibitor, clorgyline, at doses of 2 mg/Kg delivered everysecond day, did not increase the number of surviving dSNC neurons afterMPTP-induced damage. A comparison of the known molecular structures ofL-deprenyl and clorgyline (See FIG. 1), reveals that the compounds havethe same structure in the terminal portion which contains the propargylgroup (See box in FIG. 1). In contrast, the phenol ring includes the twobulky chlorines and an oxygen-linked 3 carbon chain attaches thechlorine-substituted phenol to the nitrogen with 2 carbons with a methylside chain in L-deprenyl. The inability of clorgyline to rescue the DSNneurons may relate to the chlorines preventing the propargyl group fromreaching an attachment site or may indicate that the critical structureincludes the portion of the molecule linking the phenol ring to thenitrogen.

The MAO-B inhibitor N-(2-aminoethyl)-4-chlorobenzamide hydrochloride wasfound not to rescue immature axotomized motoneurons. The compound doesnot have the triple bond C ending of the propargyl terminus of deprenyland pargyline so it appears that it attaches to a different part of theflavine portion of MAO-B.

The (+) isomer of deprenyl at a dosage of 0.01 mg/Kg was found not torescue immature axotomized motoneurons. Thus, the optical rotation ofthe terminal propargyl group relative to the phenol ring andintermediate portions of the compounds may also be important for therescue.

As discussed above, the present invention relates to the use of deprenylor derivatives or analogues of deprenyl to rescue damaged nerve cells,to pharmaceutical compositions containing deprenyl adapted for such use;and, to methods for the treatment of disorders of the nervous system byrescuing damaged nerve cells.

Deprenyl, also known as selegiline, preferably L-deprenyl (see The MerckIndex, 11th ed. 2893), derivatives of deprenyl, preferablypharmaceutically acceptable salts and esters of deprenyl; or, analoguesof deprenyl, preferably structural analogues of deprenyl or functionalanalogues of deprenyl such as pargyline, AGN-1133, AGN-1135 and MD240928, or other agents which may or may not inhibit MAO-B such asimipramine, chlorpromazine, amitriptyline,(-)2,3-dichloro-α-methylbenzylamine, N-cyclopropyl-substitutedarylalkylamines, may be used in the present invention. Most preferably,L-deprenyl and pargyline are used in the pharmaceutical compositions,therapeutic uses and methods of the present invention.

The administration of deprenyl or derivatives or analogues of deprenylmay rescue damaged nerve cells in an animal, and thus may be used forthe treatment of neurodegenerative and neuromuscular diseases and inacute damage to nervous tissue due to hypoxia, hypoglycemia, ischemicstroke or trauma. It may also be used to slow the progressive loss ofneurons associated with brain aging; although the present inventors haveshown that deprenyl does not prevent age-related death of mouse DSNneurons. More specifically, deprenyl may be used to treat Parkinson'sdisease, ALS, head trauma or spinal cord damage, patients immediatelyfollowing an ischemic stroke, hypoxia due to ventilatory deficiency,drowning, prolonged convulsion, cardiac arrest, carbon monoxideexposure, exposure to toxins, or viral infections. Deprenyl orderivatives or analogues of deprenyl may also be used to stimulatemuscle reinnervation in traumatic and non-traumatic peripheral nervedamage.

The pharmaceutical compositions of the invention contain deprenyl orderivatives or analogues of deprenyl, either alone or together withother active substances. Such. pharmaceutical compositions can be fororal, topical, rectal, parenteral, local, inhalant or intracerebral use.They are therefore in solid or semisolid form, for example pills,tablets, creams, gelatin capsules, capsules, suppositories, soft gelatincapsules, gels, membranes, tubelets. For parenteral and intracerebraluses, those forms for intramuscular or subcutaneous administration canbe used, or forms for infusion or intravenous or intracerebral injectioncan be used, and can therefore be prepared as solutions of the activecompounds or as powders of the active compounds to be mixed with one ormore pharmaceutically acceptable excipients or diluents, suitable forthe aforesaid uses and with an osmolarity which is compatible with thephysiological fluids. For local use, those preparations in the form ofcreams or ointments for topical use or in the form of sprays should beconsidered; for inhalant uses, preparations in the form of sprays, forexample nose sprays, should be considered.

The preparations of the invention can be intended for administration tohumans or animals. They contain preferably between about 1 and 4 mg ofactive component in the case of solutions, sprays, ointments and creamsand between about 0.1% and 5% and preferably between about 0.1% and 10%of active compound in the case of solid form preparations. Dosages to beadministered depend on individual needs, on the desired effect and onthe chosen route of administration, but daily dosages to humans bysubcutaneous, intramuscular or intracerebral injection generally varybetween 0.1 and 10 mg of active substance per day, preferably between 1to 10 mg per day, most preferably between 5 and 10 mg per day.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit notexclusively, solutions of the deprenyl or derivatives or analogues ofdeprenyl in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids.

As hereinbefore mentioned the invention also relates to methods fortesting a drug for activity in rescuing damaged nerve cells in apatient.

In accordance with one embodiment of the invention a method for testinga drug for activity in rescuing damaged nerve cells in a patient isprovided comprising administering an agent having neurotoxic activity toa test animal; administering the drug to the test animal; sacrificingthe test animal within a period of 20 days from completion ofadministration of the agent; taking serial sections of the brain throughthe substantia nigra compacta; determining the number of tyrosinehydroxlyase positive somata in alternate serial sections of the brainthrough the substantia nigra compacta and determining the number ofNissl stained positive substantia nigra compacta in intervening serialsections; and comparing the number of tyrosine hydroxlyase positivesomata and Nissl stained somata determined with the number of tyrosinehydroxlyase positive somata and Nissl stained somata in serial sectionsof the brain through the substantia nigra compacta of a control animalwhich has not been administered the drug.

In a preferred embodiment the agent having neurotoxic activity is MPTP,most preferably in an amount of 30 mg/kg/d, which is administered i.p.to 8 week old mice, preferably C57BL mice, for five consecutive days(days -5 to 0; total preferred cumulative dose of 150 mg/kg). Three daysfollowing cessation of MPTP administration (day 0), treatment withsaline (control animal) or the appropriate doses of the drug (testanimals) is commenced. Preferably the administration of the drug iswithheld until day 3 to ensure that all mice are exposed to comparablelevels of MPP+ and that all MPTP and its metabolites have beeneliminated from the central nervous system. Mice are killed byanaesthetic overdose (pentobarbital) followed by paraformaldehydeperfusion 20 days following their last MPTP injection. Brains arebisected longitudinally along the madline and the half brains are gluedtogether using Tissue-Tek so that surface landmarks are in longitudinalregister. The glued brains are frozen in -70° C. methylbutane and 10 μmserial sections are cut through the entire longitudinal length of eachSNc.

The number of tyrosine hydroxlyase positive somata in serial sections ofthe brain through the substantia nigra compacta may preferably bedetermined using a polyclonal TH antibody as the primary antibody and astandard avidin-biotin reaction with diaminobenzidine (DAB) as thechromogen for visualization as generally described in Seniuk, N. A. etal. Brain Res. 527, 7-20 (1990) and Tatton, W. G. et al. Brain Res. 527,21-32 (1990) which are incorporated herein by reference, and modified asfollows. Slide-mounted sections are incubated with unlabelled primary THantisera in 0.2% Triton/0.1 M phosphate buffer at 4° C. overnight.Tissues are washed with phosphate buffer then incubated for 1 hour withbiotinylated goat anti-rabbit IgG secondary antibody followed byavidin-HRP incubation. A 0.05% solution of DAB in 0.01% hydrogenperoxide is used to visualize the immunoreactive somata.

Intervening sections may be Nissl stained to define nuclear outlinesfollowing the procedure set forth in Seniuk et al., Brain Res. 527: 7,1990 and Tatton et al. Brain Res. 527:21, 1990 which are incorporatedherein by reference.

In accordance with another embodiment of the invention a method fortesting a drug for activity in rescuing damaged nerve cells in a patientis provided comprising carrying out an axotomy on a test animal;administering the drug to the test animal; determining the number ofcholine acetyl transferase positive somata for histological sectionsfrom the site of the axotomy; and comparing the number of choline acetyltransferase positive somata determined with a number of choline acetyltransferase positive somata in serial sections from the site of theaxotomy in a control animal which has not been administered the drug.

Preferably a unilateral facial nerve transection is carried out on thetest animal, preferably a rat, and paired lesion and no lesion groupsare treated with saline (control animal) or appropriate doses of thedrug (test animal). The rats are sacrificed at 21 days after axotomy andserial coronal histological sections of the brainstem at the level ofthe facial nuclei are processed for choline acetyl transferase (CHAT)immunocytochemistry using the procedure of Tatton W. G.et al, Brain Res.527:21, 1990, which is incorporated herein by reference.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES

Example 1

This example demonstrates the loss of tyrosine hydroxylaseimmunopositive (TH+) neurons from the substantia nigra compacta (SNc)following the administration of MPTP and their rescue by deprenyl.

In the first part of the study, the time course of MPTP induced neuronaldeath was established as follows. MPTP (30 mg/kg/d) was administeredi.p. to 8 week old isogenic C57BL mice (from the National Institutes ofAging colony at Jackson Laboratories, USA (C57BL/NNia)); (n=6/timeperiod) for five consecutive days (total cumulative dose of 150 mg/kg).Mice were killed by anaesthetic overdose (pentobarbital) followed byperfusion with isotonic saline (containing 5% rheomacrodex and 0.008%xylocane) and 4% paraformaldehyde 5, 10, 15, 20, 37 and 60 daysfollowing their last MPTP injection. Dissected brains were immersed in4% paraformaldehyde in 0.1 m phosphate buffer overnight and placed in20% sucrose.

In the second part of the study, the rescue by deprenyl of TH+ SNcneurons from MPTP induced loss was demonstrated as follows. MPTP (30mg/kg/d) was administered i.p. to 8 week old C57BL mice (n=6-8/treatmentgroup) for five consecutive days (days -5 to 0; total cumulative dose of150 mg/kg). Three days following cessation of MPTP administration (day0), mice were treated with saline, deprenyl (Deprenyl Canada) (0.01,0.25 or 10 mg/kg i.p.) or Clorgiline (Sigma Chemical Company, U.S.A.) (2mg/kg) three times per week. Deprenyl administration was withheld untilday 3 to ensure that all mice were exposed to comparable levels of MPP+and that all MPTP and its metabolites had been eliminated from thecentral nervous system. Doses of deprenyl were chosen to reflect thoseused in studies demonstrating that deprenyl can prolong the lifespan ofthe rat and inhibit MAO-B activity by approximately 75% but have noeffect on MAO-B activity (0.25 mg/kg) or cause inhibition of both MAO-Band MAO-A (10 mg/kg) (Knoll, J. Mt. Sinai J. Med. 55, 67-74 (1988) andKnoll, J. Mech. Ageing Dev. 46, 237-262 (1988), Demarest, K. T. andAzzarg A. J. In: Monoamine Oxidase: Structure, Function and AlteredFunction (T. P. Singer, R. W. Von Korff, D. L. Murphy (Eds)), AcademicPress, New York (1979) p. 423-430). A dose of 0.01 mg/kg deprenyl wasalso chosen; at this dose less than 10⁻⁷ M will reach the brain tissue.As a further control, mice were treated with only deprenyl and were notadministered MPTP. Mice were killed by anaesthetic overdose(pentobarbital) followed by paraformaldehyde perfusion 20 days followingtheir last MPTP injection.

For both parts of the study, brains were bisected longitudinally alongthe midline and the half brains were glued together using Tissue-Tek sothat surface landmarks were in longitudinal register. The glued brainswere frozen in -70° C. methylbutane and then 10 μm serial sections werecut through the entire longitudinal length of each SNc.

Alternate sections were processed for TH immunocytochemistry using apolyclonal TH antibody as the primary antibody and a standardavidin-biotin reaction (ABC kit, Vector Labs) with diaminobenzidine(DAB) as the chromogen for visualization as generally described inSeniuk, N. A. et al. Brain Res. 527, 7-20 (1990) and Tatton, W. G. etal. Brain Res. 527, 21-32 (1990) which are incorporated herein byreference, and modified as follows. Slide-mounted sections wereincubated with unlabelled primary TH antisera (Eugene Tech) in 0.2%Triton/0.1M phosphate buffer at 4° C. overnight. Tissues were washedwith phosphate buffer then incubated for 1 hour with biotinylated goatanti-rabbit IgG secondary antibody. followed by avidin-HRP incubation. A0.05% solution of DAB in 0.01% hydrogen peroxide was used to visualizethe immunoreactive somata. For comparative optical density measurements,sections from control and MPTP-treated brains were mounted on the sameslide to reduce the effect of slide to slide variability in the assayprocedure and were processed for immunocytochemistry.

The number of TH+ SNc neurons was obtained by counts of number codedalternate serial sections through each entire nucleus. Sections wererecounted by multiple blind observers to check any observer bias. Thevalues were corrected for section thickness (Konigsmark, B. W. In:Nauta, W. H., Ebesson S. O. E. ed Contemporary Research Methods inNeuroanatomy, New York, Springer Verlag, p. 315-380, 1970). The meanvalue plus or minus the standard error of the mean was computed for thesaline injected control mice. Subsequent data was then expressed as apercentage of this mean number as shown in FIG. 2.

Intervening sections were Nissl stained to define nuclear outlines (SeeSeniuk et al., Brain Res. 527:7, 1990; Tatton et al. Brain Res. 527:21,1990 which are incorporated herein by reference). The paired halfsections for the glued half brains insured that any differences inneuronal numbers in the experimental and control groups were not due todifferent penetration or exposure to the antibodies or the reagents.

On 20 randomly-chosen half sections through the length of each nucleusfor each animal, the region containing TH+ somata was traced using acamera lucida attachment to the microscope and the outline was thentransposed to the immediately adjacent Nissl section using localhistological features for landmarks (each nucleus usually included about90 pairs of sections). The numbers of Nissl somata containing anucleolus within the outline were counted according to three size groups(small--140 to 280 μm², medium--300 to 540 μm² and large--540 to 840μm²), excluding glial profiles (40 to 100 μm²), using criteria similarto those of the rat SNc (Poirier et al. 1983 Brain Res. Bull. 11:371).Numbers of TH+ somata were plotted against numbers of Nissl somata forthe corresponding areas of 20 immediately adjacent sections. The jointNissl/TH+ counts provide a means for determining whether reductions inthe numbers of TH+ SNc somata are due to neuronal destruction or a lossof TH immunoreactivity by surviving neurons (see Seniuk et al. 1990,supra, for details as to rationale for the procedure).

FIG. 3 shows a loss of TH+ somata from the SNc from days 0 to 20 postMPTP, with no decline thereafter. 20 to 30% of TH+ somata were lost byfive days after completion of the injection schedule (day 5); loss ofTH+ neurons continued over the next ten to fifteen days with no furtherdisappearance thereafter. This continual loss of TH+ neurons could notbe accounted for by the presence of MPTP or its toxic metabolite MPP+,due to its rapid elimination from the body (Johannessen, J. N. et al.,Life Sci, 36,219-224 (1985); Markey, S. P. et al., Nature, 311, 465-467(1984); and Lau et al., Life Sci. 43, 1459-1464 (1988)). Some neuronshave the capacity to initiate repair following axonal damage, such asthat seen with MPTP, by reactivating DNA transcription "programs"similar to those utilized by developing neurons to extend their axons orneurites (see Barron, K. V. in Nerve, Organ and Tissue Regeneration:Research Perspectives (eds. Seil, F. J.), 3-38 (Academic Press, NewYork, 1986). In the case of the TH+ SNc neurons, it would appear that acritical 20 day period exists in which these neurons either undergoeffective repair and recovery following MPTP-induced damage or they die.

Joint plots of the counts of TH+ and Nissl stained SNc somata fromcorresponding areas of immediately adjacent sections in mice treatedwith saline only (values for three animals are pooled in FIGS. 3A1-A3)show that the numbers of TH+ somata are linearly related to the numberof Nissl somata and are closely scattered around an equal value diagonal(illustrated by the diagonal lines in FIG. 3) for the medium-sized SNcsomata (FIG. 3A2)) and the large-sized SNc somata (FIG. 3A3). In eachplot in FIG. 3, the mean ±1.0 standard deviation for the Nissl countsand the TH+ counts of somata per half section are shown at the upper endof each Y axis and the right end of each X axis respectively. For themedium and large somata the mean number of Nissl somata exceed thecorresponding mean number of TH+ somata by 5-10% which appears tocorrespond to the percentage of nigrostriatal neurons which are not TH+(Van der Kooy et al. Neuroscience 28:189, 1981).

Joint counts of the small-sized SNc somata in the saline treated animalsshow that only a small proportion of the small neurons are THimmunoreactive and therefore dopaminergic (FIG. 3A1). These results arein keeping with previous findings in rodents which indicate that thelarge and medium sized somata are those of dopaminergic nigrostriatalneurons while the smaller somata are largely those of locally ramifyinginterneurons (Van der Kooy et al., 1981 supra; Poirier et al. Brain Res.Bull. 11:371, 1983). Joint Nissl/TH counts of somata in the animalstreated with MPTP alone or MPTP followed by saline (values for threeMPTP-saline animals are pooled in FIGS. 3B1, 3B2 and 3B3) confirmed thatby 20 days after the completion of the MPTP treatment the loss of TH+somata represented the death of SNc neurons rather than a loss of THimmunoreactivity in surviving neurons. FIGS. 3B2 and 3B3 show that eventhough the counts of Nissl and TH+ somata are reduced from 21.6±15.5 and20.6±15.5 per section to 12.4±8.0 and 11.4±7.2 for the mediumsized andlarge-sized somata respectfully (values are means ±1.0 standarddeviation), the almost equal value relationships between the counts weremaintained. If the SNc neurons were losing TH immunoreactivity but notdying, the scatter of the joint plots would be expected to shift to lociabove the equal value diagonal (Seniuk et al. Brain Res. 527:7, 1990).Furthermore, FIG. 3B1 shows that the numbers of small-sized Nisslstained somata decreased slightly (26.2±18.3 to 22.4±12.5 per section)in accord with the reduction (4.1±2.8 to 2.3±1.6 per section) in the TH+component of the small-sized SNc somata. If some of the losses of mediumand large sized SNc somata were due to atrophy so that theircross-sectional areas no longer fell within the medium and large sizeranges in response to the MPTP treatment, one would expect an increasein the numbers of small sized Nissl stained somata.

Joint Nissl/TH+ plots for days 0, 3, 5, 10, 15 and 20 after completionof the MPTP treatment and for the saline controls are shown in FIGS. 4and 5.

FIG. 4 represents Nissl/TH plots for the three major size groups of SNcsomata in rodents (small cross sectional somal areas, 140-280 μm²,medium cross sectional somal areas, 300-540 μm² and large crosssectional somal areas, 540-840 μm²) for the saline control animals. Thedata was pooled for saline controls sacrificed at days 0, 3, 5, 10, 15and 20 after completion of the MPTP exposure. As previously shown, theJoint Nissl/TH+ plots for the small SNc somata largely fall above theequal value diagonal (mean values of 31.9±19.2 per section for Nisslcounts and 3.5±2.6 for TH+ counts) since most of the small somata arethose of non-dopaminergic neurons. In contrast, the medium and largesomata which are known to be largely dopaminergic cluster closely aboutthe equal value diagonal (Nissl mean/section of 15.8±12.8 and TH+mean/section of 14.7±12.3 for the medium-sized somata and Nisslmean/section of 3.2±3.5 and TH+ mean/section of 2.9±2.4 for thelarge-sized somata). Hence for the saline controls the great majority ofmedium sized and large-sized Nissl stainable somata are also THimmunoreactive.

FIG. 5 shows that at Day 0 (the final day of the MPTP exposure), a majorproportion of plots for the medium-sized somata (medium-sized somataaccount for more than 90% of the dSNc neurons) fall above the equalvalue diagonal and above the range of the points established for thesaline treated animals. This indicates that a significant proportion ofthe medium-sized dSNc neurons have lost detectable TH immunoreactivitybut have not yet died at Day 0 (compare the mean Nissl counts/sectionfor the pooled saline controls of 15.8±12.8 to that for the Day 0 MPTPexposed of 14.8±9.7 showing that 14.8/15.8 of the medium sized somataare still present at Day 0). Gradually for days 5 through 20 the locusof the points return to within the band established for the salinecontrols while the extent of the points along the equal value diagonalshrinks toward the origin of the plots. That progressive change in theloci of the points in the joint Nissl/TH+ plots indicates that theneurons are gradually dying over the 20 day period so that by day 20 allof the surviving medium-sized neurons have detectable THimmunoreactivity.

FIG. 6 shows superimposed plots for the percentage of Nissl stainedsomata and the percentage of TH immunoreactive somata relative to themean values for the saline controls for Day 0 through Day 60. Thedifference between the TH immunoreactive percentages and theNissl-stained percentages demonstrates the percentage of dSNc neuronswhich are sufficiently damaged to suspend TH synthesis but have not dieddue to the toxin. Hence at Day 3, when the deprenyl treatment wasinitiated, an average of 37% of the dSNc somata had lost detectable THimmunoreactivity but only 4% had died. The two plots converge betweendays 15 and 20 when the percentage of TH immunoreactive somata is notdifferent from the number of Nissl stainable SNc somata. The differencebetween the two plots can be taken to estimate the percentage ofseverely damaged dSNc neurons that are potentially rescuable at eachtime point after MPTP exposure.

According to the superimposed plots in FIG. 6, 84% of the dSNc neuronsthat died by days 15-20 could potentially be rescued at Day 3. Hence,since it was found that deprenyl rescued 66% of that 84%, deprenyltreatment in fact rescued 79% of the neurons that had not died beforetherapy was initiated.

FIG. 7 presents the raw counts of TH+ SNc somata for individual SNcnuclei taken from alternate 10 micron serial sections throughout theentire rostro-caudal length of each nucleus and expressed as acumulative frequency distribution. Four representative trials for eachtreatment are presented in FIG. 7. Values for neuronal counts from micetreated with saline alone, MPTP (150 mg/kg) and saline and MPTP plusdeprenyl (0.25 mg/kg, 3 times per week) are shared with those presentedin histogram fashion in FIG. 8. As shown in FIG. 8, the cumulativefrequency distribution curves for all SNc nuclei (n=4/treatment group)have a similar pattern indicating that the loss of TH+ somata followingMPTP and their rescue by deprenyl occurred in all parts of the nucleialthough it appears to be greatest in the rostral portion of the nuclei(sections 10-40) that contains neurons which are relatively moreresistant to the toxin. FIG. 8 also illustrates that there is no overlapin individual frequency distribution curves between the three groups ofanimals.

FIG. 9 shows TH+ somal counts for dSNC neurons along the rostrocaudallength of a nucleus. Rostrocaudal counts for 6 animals are superimposedin each panel. The area under each represents the total number ofimmunoreactive dSNC neurons and it shows the rescue by deprenyl.

Data shown in FIG. 8 represent the average number for all trials (n=6-8mice/treatment group, i.e. 12-16 SNc nuclei) ±S.E.M. of TH+ somata/SNcnucleus. To obtain these values, raw counts of TH+ somata were convertedto neuronal numbers using a correction factor of 2.15 as described byKonigsmark, B. W., in Contemporary Research Methods in Neuroanatomy(eds. Nauta, W. H. and Ebesson SOE) 315-380 (Springer Verlag, New York,1970). FIG. 8 shows an increased number of TH+ SNc somata in thedeprenyl treated mice relative to animals receiving MPTP alone,suggesting that deprenyl prevented a portion of the neuronal lossassociated with MPTP-induced toxicity. Both low and high doses ofdeprenyl were equipotent in preventing the TH+ SNc neuronal loss.

In particular FIG. 8 shows that the mean corrected numbers of TH+ somatafound for animals treated with saline only of 3014±304 (mean±SEM) weresignificantly reduced (Mann-Whitney Test, p<0.001) in the animalstreated with MPTP only (1756±161) and the MPTP-Saline groups (1872±187,1904±308 and 1805±185). Therefore MPTP caused average losses of 36, 38and 42% of TH+ somata in those three MPTP pretreated groups (black barsin FIG. 8). All the MPTP saline control groups are statistically thesame (p>0.05). FIG. 8 also shows that Clorgiline an MAO-A inhibitor doesnot rescue the neurons since the MPTP-Saline (1706±155) andMPTP-Clorgyline (1725±213.6) values are statistically the same.

Deprenyl significantly increased (p<0.005) the number of TH+ SNc somataafter MPTP to 2586±161 (14% loss), 2535±169 (16% loss) and 2747±145 forthe 10, 0.25 and 0.01 mg/kg doses respectively. Hence all doses ofdeprenyl reduced the loss of TH+ somata caused by the MPTP to less than50% of the loss that was found when the MPTP was followed by saline i.e.all three deprenyl doses produce similar and statistically significant(p<0.001) increases in neuronal numbers compared to the saline treatedanimals.

FIG. 10 also shows the mean corrected number of TH+ somata found foranimals treated with saline only, MPTP only, MPTP-saline,MPTP-clorgyline, MPTP-deprenyl with a table illustrating the timing ofthe various treatments. It also shows somal counts for animals onlytreated with deprenyl. Deprenyl alone does not alter the counts of TH+somata in animals not previously exposed to MPTP.

The results illustrated in FIGS. 7 and 8 are even more striking when oneconsiders the time-course of MPTP-induced loss of TH+ SNc neuronsdiscussed above. By day five 75% of the TH+ SNc neurons which would dieby day twenty had already lost their TH-immunoreactivity and only 25% ofthe TH+ SNc neurons which would die continued to looseTH-immunoreactivity between days 5 and 20. Assuming that the time courseof neuronal loss was identical in the first and second part of thestudy, the numbers of TH+ SNc somata would have decreased from a mean of3014 somata/nucleus to 2169 at day 3 and then further declined to anaverage of 1872 somata/nucleus by day 20. Deprenyl-treated mice (0.25mg/kg) had an average of 2535 somata/nucleus thereby showing thatdeprenyl rescued all TH+ SNc neurons that would have died during the 17days of administration and may even have rescued some TH+ SNc neuronswhich were no longer identifiable by TH+ immunocytochemistry.

The Joint Nissl/TH+ counts in FIGS. 3C1-C3 were plotted for pooled datafrom three animals treated with MPTP followed by 0.25 mg/kg doses ofdeprenyl. FIG. 3C2 shows a joint reduction in the loss of Nissl and TH+medium-sized SNc somata compared to that for the MPTP-saline animals(FIG. 3B2). There is a relatively smaller reduction in the loss oflarge-sized somata for the MPTP-deprenyl animals (FIG. 3C3) compared tothat for the MPTP-saline animals (FIG. 3B3). The joint Nissl/TH+ plotsestablish that reduced loss of TH+ SNc somata in the MPTP-deprenyltreated mice is due to reduction in neuronal death rather than areduction in the number of neurons which are not TH immunoreactive.

Example 2

MPTP- Mice were administered deprenyl (0.01 mg/dg or 0.25 mg/kg)following the procedure set out in Example 1. MAO-A and MAO-Bmeasurements were obtained in accordance with the method set out below24 hours after the first 0.25 mg/kg or 0.01 mg/kg deprenyladministration and 18 days later (corresponding to day 21 which would bejust after the animals were sacrificed for the immunochemistry at day20).

MAO activity was assayed in fresh tissue homogenates by the method ofWurtman, R. J. and Axelrod, J., (Biochem Pharmacol 1963;12:1439-1444),with a modification of substrates in order to distinguish between MAO-A-and MAO-B. This method relies on the extraction of acidic metabolites ofeither (14-C)-serotonin (for MAO-A) or (14-C) phenylethylamine (forMAO-B) in toluene/ethyl acetate. Tissue homogenates were incubated inpotassium phosphate buffer containing either radiolabelled serotonin(100 micromolar) or phenylethylamine (12.5 micromolar) for 30 minutes at37° C. The reaction was stopped by the addition of HCl and acidmetabolites extracted into toluene/ethyl acetate. Radioactivity in thetoluene/ethyl acetate layer is determined by liquid scintillationspectrometry. Blanks are obtained from either boiled tissue homogenatesor form reaction mixtures containing enzyme (Crane, S. B. and Greenwood,C. E. Dietary Fat Source Influences Mitochondrial Monoamine OxidaseActivity and Macronutrient Selection in Rats. Pharmacol Biochem Behav1987;27:1-6).

FIG. 11 presents the MAO-A and MAO-B measurements for 24 hours after thefirst 0.25 mg/kg or 0.01 mg/kg and 18 days later (corresponding to day21 which would be just after the animals were sacrificed for theimmunocytochemistry at day 20). Hence since MAO-B inhibition (100%--MAO-B activity) would gradually increase over the 17 day treatmentperiod, the two measures (labelled d4 and d22 to correspond to FIG. 2)give a picture of MAO-A and MAO-B activity at the beginning and end ofthe treatment period.

The KS probability shown in the brackets above each pair (saline anddeprenyl treatment) represents the results of the Kolmogorov-Smirnov twosample non-parametric statistical testing (Siegel, S. Non ParametricStatistics for the Behavioral Sciences, McGraw-Hill Book Company, NewYork, 1956, pp. 127-136) to determine if the deprenyl-saline pairs aredrawn from the same population. The probability value indicates theprobability that the data comes from the same population. A value ofp<0.5 is required to detect any significant differences and p<0.01 ispreferable. Hence there is weak but detectable inhibition of MAO-A at d4for the 0.25 mg/kg deprenyl dose which may be real since the MAO-Binhibitor may cause weak MAO-A inhibition at higher doses. The 0.25rag/dose causes strong MAO-B inhibition at both d4 (72% activity, 28%inhibition) and d22 (31% activity, 69% inhibition). Ninety percent ormore MAO-inhibition was required for anti-depressant effects butconceivably 28 to 69% MAO-B inhibition might mediate the rescue atdeprenyl doses of 0.25 mg/kg.

Most importantly, the 0.01 mg/kg dose did not produce any significantMAO-A or MAO-B inhibition at d4 and d22. Hence the marked rescue with0.01 mg/kg is equipotent to that with 0.25 mg/kg but cannot be due toMAO-B inhibition. Therefore, deprenyl may activate a receptor through a3D structure which may not be related to the structure which blocksMAO-B.

Example 3

Male, C57BL/6J mice obtained at five weeks of age from Jackson Labs (BarHarbour, Me.) were housed in individual cages and allowed food and waterad libitum. Mice were given an initial two week acclimatization periodto a 12:12 hour light:dark (LD) cycle in an isolated room kept at aconstant temperature of 21° C. Subjective `day` began at 8:00 hourswhile subjective `night` began at 20:00 hours. Light levels weremaintained at 200 lux during the subjective day. Locomotory movementswere selectively quantified with a Stoelting Electronic ActivityMonitor, individual sensor boxes being placed under each cage. Higherfrequency signal interruption such as feeding or grooming events werenot recorded. Locomotory movements for individual mice were continuouslymonitored under continual darkness (DD) or under LD conditions for 90 to120 days. After approximately 20 days the mice were treated with twicedaily injections for 5 days (pre injection days -5 to 0) of saline orMPTP (to achieve cumulative doses of 37.5, 75, 150 and 300 mg/kg).Injections were always given during the subjective day, the firstinjection occurring 4 hours after `lights on` and the second, 4 hoursbefore `lights off`.

Spectral analysis (Bloomfield, P. Fourier Analysis of Time Series: AnIntroduction; John Wylie and Sons: New York, 1976, Brigham, E. O. TheFast Fourier Transform; Prentice-Hall, New York, 1974, Marmarelis, P.Z.; Marmarelis, V. Z. Analysis of Physiological Systems--The White-NoiseApproach; Plenum Press: New York and London, 1978) of the locomotoryactivity was done with a SYSTAT statistical software program using fastFourier transforms. Activity counts from periods just exceeding 240hours (about 10 days) or 120 hours (about 5 days) were used. The numberof samples were chosen to Just exceed 128 or 256 in order to fulfil therule of powers of 2. Before Fourier decomposition the activity valueswere treated with a split-cosine-bell taper to reduce leakage fromstrong components into other components. These values were then paddedwith zeros to 512 samples. The mean was then removed from these valuesand the Fourier transform was calculated for 100 lags to encompasshours/cycle values of 5.12 to 512. The magnitudes were squared todetermine the power of each component and the power for each hour/cyclevalue was expressed as a percentage of the total power.

Neurochemical assays were performed at 5, 10, 15 and 20 days followingthe last of the MPTP injections. The mice were sacrificed by cervicaldislocation and the brain removed. Striatal tissue was dissected so asto include the nucleus accumbens and the caudate. The tissue was frozenin 2-methylbutane (Kodak) at -70° C. until their catecholamineconcentrations were measured by reverse-phase ion-pair high performanceliquid chromatography (HPLC) with electrochemical detection. Tissuesamples were weighed, then homogenized in 0.2N perchloric acidcontaining dihydroxybenzylamine as internal standard and extracted ontoalumina (Mefford, I.N.J. Neurosci. Meth. 1981, 3, 207-224). Thecatecholamines were desorbed into 0.1N phosphoric acid, filtered andinjected onto an Ultrasphere 0DS 5 um column. The mobile phase contained7.1 g/1 Na2HPO4, 50 mg/1 EDTA, 100 mg/1 sodium octyl sulphate and 10%methanol. The detector potential was 0.72 versus a Ag-AgCl referenceelectrode. Interrun variability was approximately 5%.

FIG. 12 shows 92 days of typical recording and the black bar indicatesthe interval of MPTP injection 150 mg/kg in total, 30 mg/kg daily forfive days). Each vertical bar on the activity trace represents the sumof activity for one hour. Note that there is a slower rhythm with aperiod between 100-200 hours superimposed on a faster (about 24 hour)circadian rhythm which introduces a cyclic variation into the amplitudeof the activity peaks. The regularity of these patterns, as well as theamplitude of activity, was significantly affected during the MPTPinjection period (675 h-842 h), but seemed to "recover" by 1200 hours,viz. between days 15-20 post-injection.

Analysis of the locomotory activity in the time domain was complicatedby the superimposition of multiple endogenous activity cycles so thatFourier analysis was used to quantitate the data. High resolution powerspectra for LD and DD preinjection control periods from a salineinjected mouse are shown in FIG. 13. The spectra were calculated for 256activity counts then padded to 4096 values with zeros before the Fouriertransform was applied. In FIG. 13A, both LD and DD spectra display amajor peak at approximately 24 hours/cycle which includes in excess of75% of total power. Note the slight shift in the centroid of the DD peakto a cycle length which is approximately 9 minutes shorter than the LDpeak. In FIG. 13B a secondary peak occurs between 100-250 hours/cyclewhich is consistent with previous observations from the raw data of FIG.12. This peak is shifted by about 50 hours/cycle for the DD spectra ascompared to the LD spectra. Longer hours/cycle values did not reveal anyother peaks. Note that a third smaller peak arising only during LDentrainment occurs over 60-90 hours/cycle. The clear separation of thecircadian peak from the slower peaks made it possible to independentlyevaluate the changes in the power of the dominant 24 hour componentafter MPTP treatment. The locomotory activity was therefore measured asthe percentage power under the 22-26 hours/cycle peak.

In FIG. 14, panel A shows that interruption of the animals' endogenousactivity by saline injections was sufficient to reduce the percentagepower of the P22-26 relative to pre-inJection and post-injection days.Hence, activity changes like those in Panel B could not be reliablyinterpreted for the MPTP injection period. Saline injections did notproduce any changes in the P22-26 in the post-injection period (Panel Cfor an example). In contrast, the 150 and 300 mg/kg doses (see FIG. 15)resulted in marked depression of the P22-26 which recovered by days 12to 20 (Panels B and D).

FIG. 15 shows that saline and 37.5 or 75 mg/kg MPTP injections did notalter P22-26 locomotory activity significantly from that of the controlpre-injection days (the error bar represents ±1 s.d. for the pooledcontrol activity). In contrast, peak power for the P22-26 was reduced to20-60% of mean control values in the 5 days following 150 or 300 mg/kgMPTP treatment and returned to normal by median Day 20.

A second series of animals, treated with 150 mg/kg MPTP or saline, weresacrificed for TH immunocytochemistry and sections were visualized withavidin-conjugated horseradish peroxidase and diaminobenzidine at days 5,10, 15, 20 and 60 following MPTP injection. The paraformaldehydeperfused brains were bisected along the midline and halves from asaline-injected and an MPTP-injected animal were glued together usingTissue-Tek so that surface landmarks were longitudinally in register.Ser. 10 μm sections were taken through the brainstem to encompass an SNcfrom both animals so that SNc neurons from the saline and MPTP animalswere immediately adjacent and were exposed to similar concentrations ofthe antibodies and reagents. Panel A and Panel B (FIG. 16) present SNcsections for glued brains at Days 5 and 20.

FIG. 17, Panel A presents the counts of TH+ SNc and VTA neuronal somatafollowing MPTP treatment taken through whole nuclei expressed as apercentage of the mean counts for the corresponding saline-injectedanimals (error bars are s.d.). MPTP injected animals are represented bythe filled symbols. Note the gradual decrease in the number of SNcsomata with detectable TH immunoreactivity from Days 5 to 20 with anapparent maintenance of the number of TH+ somata after Day 20. Panels B,C and D present the concentration of striatal DA and DOPAC for thesaline and MPTP injected animals. Note the similarity of the time coursefor the recovery of striatal DA concentrations toward normal levels withrecovery of locomotory activity in FIG. 15. The DOPAC/DA ratio shows amarked increase and rapid decline over Days 5-10 for the MPTP injectedanimals and then maintains a constant Level at about 2 times that of thesaline injected animals.

A computer optical density (OD) system was used to measure somalcytoplasmic TH immunoreactivity and the background immunoreactivity inthe immediately adjacent tissue for randomly chosen SNc and VTA somata(Tatton, W. G. et al. Brain Res. 1990, 527, 21-32) for the glued brainsections. Background OD per unit area was subtracted from somal OD perunit area for each cell to obtain an estimate of cytoplasmic THimmunodensity per unit area. The mean background 0D for the salineinjected half of each glued section was used to normalize the values forthe MPTP background OD and the saline and MPTP cytoplasmic ODs. FIG. 17presents distributions for the normalized background and cytoplasmicmeasurements for TH+ SNc somata at Days 5-20 after saline or 150 mg/kgMPTP injections. In this and other studies using the glued brains,background values did not differ significantly (p<0.05) for the salineinjected and MPTP injected halves thereby allowing valid comparisons ofthe cytoplasmic values. The control distributions for the salineinjected animals often revealed a bimodal distribution of THimmunodensity for the SNc somata ranging from 0.5 to 6 times meanbackground levels with modes at about 2 and 4 times mean backgroundlevel.

Within 5 days there was a marked reduction in cytoplasmic THimmunodensity for the MPTP treated SNc and VTA somata with a gradualrecovery to a distribution approximately that of the saline controls by20 days post-injection (FIGS. 17 and 18). The recovery of the THimmunodensity of SNc and VTA neurons following MPTP treatment parallelsthe recovery of striatal DA concentrations and locomotory activity.

The inventors have adapted spectral analysis techniques with fastFourier transforms to the analysis of long term locomotory activity inmice treated with MPTP. This provides both highly sensitive andreproducible data that is not dependent on subjective assessment ofanimals that have been aroused by recent handling or the presence ofobservers. Initially, it was proposed that MPTP did not produce motordeficits in rodents due to the view that rat and mouse SNc neurons wereresistant to the toxin. This was based largely on neurochemical datathat reported only transient changes in striatal dopamine following MPTP(Ricuarte, G. A. et al. Brain Res. 1986, 376, 117-124, and Walters, A.,et al. Biogenic Amines 1984, 1, 297-302). Others reported slowed limbmovements, abnormal gait and chronically reduced locomotory activity inmice treated with high doses of the toxin which appeared to correlatewith maintained changes in striatal DA concentration (Duvoisin, R. C.,et al. In Recent Development in Parkinson's Disease; S. Fahn et al.Raven Press: New York, 1986; p. 147-154, Heikkila, R. E., et al. Science1984, 224, 1451-1453, Heikkila, R. E., et al. Life Sci. 1985, 36,231-236). Previous measurements of changes in locomotory activity inrodents following MPTP unfortunately have been either short term (SaghalA., et al. Neurosci. Lett. 1985, 48, 179-184) or brief isolatedmeasurements (Willis, G. L., et al. Brain Res Bull 1987, 19, 57-62). Todate there has been no satisfactory explanation of the behavioralrecovery observed in various MPTP models including the cat (Schneider,J. S., eta la. Exp Neurol 1986, 91, 293-307), the marmoset (Waters, C.M., et al. Neuroscience 1987, 23, 1025-1039) or the rodent (Chiueh, C.C., et al. Psychopharmacol. Bull. 1984, 20,548-553, and Johannessen, J.M. et al. Life Sci. 1985, 36, 219-224).

Locomotory activity as measured by the power under the P22-26 peak,striatal DA concentration and TH immunodensity in SNc and VTA somata arecorrelated in their recovery toward normal after MPTP treatment. Thenumbers of SNc and VTA somata with detectable TH immunoreactivity decayto a steady state level over the first 20 days after MPTP treatment.Hence dopamine content in the striatum is increasing while the number ofSNc and VTA neurons with detectable TH content is decreasing. The rapidrise and fall of the DOPAC/DA ratio likely is related to the death of DAterminals in the striatum with loss of DA into the extracellular space.Yet the ratio is maintained at an increased level after Day 15 insupport of the earlier findings suggesting that DA synthesis isincreased in SNc neurons surviving MPTP exposure.

The measurements of TH immunodensity in the somata of SNc and VTAneurons are unlikely to provide a linear estimate of TH concentration.Although the use of the peroxidase reaction likely provides a linearestimate of the numbers of the secondary antibody-avidin complexes inthe cytoplasm (Reis, D. J., et al. In Cytochemical Methods inNeuroanatomy Alan R. Liss, Inc.: New York, 1982; p. 205-228), theaffinity constants for the inventors' polyclonal antibodies and thosefor the immunoreaction between the primary and secondary antibodies maynot provide for a linear relationship between the concentration of theepitope and the concentration of avidin molecules. Yet, the resultsprobably do indicate a recovery in TH concentrations in the somata ofVTA and SNc surviving MPTP exposure. The recovery of TH immunodensityparallels the increases in striatal DA content which suggests that arecovery of TH synthesis is a factor in the recovery of DA content andpossibly increased DA synthesis by individual surviving neurons.

Neostriatal dopaminergic and other ctecholaminergic systems in rodentshave been related to the generation of locomotory activity (Tabar J., etal. Pharmacol Biochem Behav 1989, 33, 139-146, Oberlander, C., et al.Neurosci. Lett. 1986, 67, 113-118, Melnick, M. E. et al. 17th AnnualMeeting Of The Society For Neuroscience, New Orleans, La., USA, November1987, 13, Marek, G. J., et al. Brain Res 1990, 517, 1-7, Kostowski, W.,et al. Acta Physiol. Pol. 1982, 33, 385-388, Fink, J. S., Smith , G. P.J. Comp. Physiol. Psych. 1979, 93, 24-65). Yet the specific role, ifany, of SNc or VTA neurons is uncertain. Hence the correlated recoveriesfor SNc and striatal parameters relative to the locomotory activity donot necessarily imply cause and effect. Yet the present inventors havesuggested that since MPTP causes similar loss of TH+ neurons in avariety of catecholaminergic systems (Seniuk, N. A. et al. Brain Res.1990, 527: p.7-20), similar recovery of transmitter-related function inthose systems to that we have shown for SNc and VTA dopaminergic neurons(Seniuk, N. A. et al. Brain Res. 1990, 527: p.7-20) may underlie thebehavioral recovery. The recovery of DA synthesis may represent anattempt of the SNc neurons surviving the MPTP exposure to compensate forthe loss of their fellows an that a component of the compensation isrelated to a recovery and then increased synthesis of tyrosinehydroxylase in the neurons surviving the MPTP exposure.

Example 4

An experiment was carried out to determine whether deprenyl can reducethe death of other axonally-damaged neuronal phenotypes e.g. ratmotoneurons. The proportion of rat motoneurons which die after axotomyis maximal during the first 4 days of life (80-90% loss) and thendiminishes to adult levels (20-30% loss) over the next 3 to 4 weeks(Sendtner et al. Nature, 345, 440-441, 1990, Snider W. D. and Thanedar,S. J. Compl. Neuro 1, 270,489, 1989). Two groups (n=6) of fourteen dayold rats received a unilateral facial nerve transection (lesion) whiletwo groups were unlesioned (no lesion). Paired lesion and no lesiongroups were treated with saline, deprenyl (0.01 and 10 mg/kg), pargyline(10 mg/kg) every other day. The rats were sacrificed at 21 days afteraxotomy and serial coronal histological sections of the brainstem at thelevel of the facial nuclei processed for choline acetyl transferase(CHAT) immunocytochemistry (Tatton et al, Brain Res. 527:21, 1990 whichis incorporated herein by reference) and Nissl staining (Seniuk et al.,Brain Res. 527: 7, 1990; Tatton et al. Brain Res. 527:21, 1990 which areincorporated herein by reference) (FIG. 19).

In particular, the right facial nerves were transected at their exitsfor the stylomastoid foramen under halothane-nitrous oxide anaesthesiafor two groups of 14 day old Sprague-Dawley rats while two other groupswere unoperated (n=6 in each group). On the day of the surgery, alesioned and an unlesioned group were begun on deprenyl 10 mg/kgintraperitoneally every second day until sacrifice. The other lesionedand unlesioned groups were given identical injections with saline.Twenty one days after the transections, the rats were killed byanaesthetic overdose followed by perfusion with isotonic saline and 4%paraformaldehyde in phosphate buffer. Brains from the unlesioned groupswere bisected longitudinally along the midline and the half brains fromsaline treated and deprenyl treated animals were glued together usingTissue-Tek so that the surface landmarks coincided. The glued brains forthe unlesioned animals and the intact brains for the lesioned animalswere frozen in -70° C. methylbutane and 10 μm serial sections were cutthrough the portion of the medulla containing the facial nuclei. Everythird serial section was reacted with a polyclonal antibody against ChATthen incubated with biotinylated secondary antibody, followed byincubation with HRP conjugated avidin and finally reacted withdiaminobenzidine and H₂ O₂ (Tatton et al., Brain Res. 527:21, 1990). Thepaired sections for the glued half brains insured that any differencesin immunoreaction between the deprenyl and saline unlesioned controlgroups were not due to different penetration or exposure to theantibodies or the reagents.

The following experiments were also carried out using the proceduresdescribed above:

A group of fourteen day old rats received a unilateral facial nervetransection (lesion) while groups were unlesioned (no lesion). Pairedlesion and no lesion groups were treated with saline or deprenyl (10mg/kg) every other day. The rats were sacrificed as and ChATimmunochemistry was carried out as described herein.

A group of fourteen day old rats received a unilateral facial nervetransection and were treated with 10 mg/kg deprenyl every other day for21 days. Animals were sacrificed at 35 days of age and at 65 days of ageand ChAT immunochemistry was carried out as described herein.

A group of one day old rats received a unilateral facial nervetransection and were treated with deprenyl every other day with salineor deprenyl (10 mg/kg). The animals were sacrificed at 8 days of age andChAT immunochemistry was carried out as described herein.

FIG. 19 shows photomicrographs of adjacent ChAT immunoreacted (A1 andB1) and Nissl stained (A2 and B2) sections through the facial nucleusipsilateral to transection of the facial nerve. A1 and A2 are for salinetreated animals and B1 and B2 are for deprenyl treated animals.

FIG. 20 is a bar graph for the counts of ChAT+ somata for the facialnuclei for the different lesion and treatment groups (bars-means, errorbars--standard deviations). ChAT immunoreactive somata containingnuclear profiles were counted from every third section taken seriallythrough entire facial nuclei. The value at the top of each bar is themean. The Ipsi.Lesion and Contra.Lesion indicate the nuclei locatedipsilaterally and contralaterally to the facial nerve transectionrespectively. The counts were not adjusted to estimate the total numbersof ChAT+ somata in the facial nuclei, so the numbers for unlesionedgroups are approximately one third of values reported for counts ofNissl stained somata. The values were compared statistically in apairwise fashion using the Mann Whitney U test.

As shown in FIG. 20 counts of CHAT immunopositive (ChAT+) somata forevery third serial section through the full lengths of the facial nucleiwere statistically the same (p=0.520) for the no lesion-saline and theno lesion-deprenyl groups. In contrast, the numbers of ChAT+ somatadecreased significantly for the lesion-saline group for the facialnuclei both ipsilateral (23.8% no lesion-saline, p=0.003) andcontralateral (82.2% no lesion-saline, p=0.024) to the facial nervetransection. Deprenyl treatment more than doubled the number of ChAT+somata for the ipsilateral lesioned facial nucleus (52.7% nolesion-saline p=0.004) and prevented the decrease in the ChAT+ countsfor the contralateral nucleus so that they were statistically the sameas the no lesion groups (p=0.873).

FIG. 21 shows the joint Nissl/ChAT+ counts of adjacent sections. One ofeach pair of intervening sections between those that were immunoreactedfor CHAT was Nissl stained. With the aid of a camera lucida the numberof ChAT+ somata and Nissl-stained nucleolus-containing somata(Oppenheim, R. W. J.Comp. Neurol. 246:281, 1986 for criteria) werecounted in matching areas of adjacent sections on 20 randomly-chosensections through the length of each nucleus for each animal. Nisslcounts were then plotted against ChAT+ counts for the adjacent sections(values from three animals in each lesion-treatment group were pooled).Comparison of Nissl and ChAT+ somal counts were done to determinewhether decreases in the number of immunopositive somata reflected thedeath of the motoneurons or loss of immunoreactivity.

The joint plots of the Nissl/CHAT somal counts for the no lesion groups(FIG. 21) show distributions. that are symmetrical around the equalvalue diagonal with similar means and standard deviations for the saline(Nissl 27.6±12.04, ChAT+ 27.3±13.80, p=0.526, Nissl and CHAT counts forthe same groups were compared using the paired t test) and deprenylgroups (Nissl 28.9±13.2, CHAT 28.5±13.8, p=0.641). The ipsilaterallesion-saline animals (FIG. 21) show lower joint values with anasymmetrical distribution with respect to the equal value diagonal (theshift to higher Nissl values is marked by an arrow) with is reflected inthe higher mean value for the Nissl counts (12.6±4.18) relative to theChAT+ counts (9.7±4.0, p=0.001). The lesion-deprenyl points (FIG. 21B)showed a smaller reduction than the saline points and had a symmetricaldistribution around the equivalent value diagonal (Nissl 17.6±±6.5,ChAT+ 17.5±6.1, p=0.616). Finally, the plot for the contralateral lesionanimals (FIG. 21C) shows that the points for both the saline (Nissl24.6±10.1, ChAT+ 24.8±10.7, p=0.159) and deprenyl (Nissl 28.9±12.1,28.5±12.0, p=0.741) groups are symmetrically distributed relative to theequivalent value diagonal.

Thus, the distribution of the joint Nissl/ChAT+ plots to above the equalvalue diagonal and the significant difference between the joint Nissland ChAT+ counts for the ipsilateral lesion-saline animals (FIG. 21)showed that about 84% of the decrease in the numbers of ChAT+ somatashown in FIG. 20 resulted from motoneuronal death while loss of ChATimmunoreactivity only caused about 16% of the decrease in ChAT+motoneurons. The joint counts also showed that all of the loss of ChAT+somata from the contralateral nuclei resulted from motoneuronal death.Most importantly, the joint counts established that deprenyl treatmentcaused a marked reduction in the motoneuronal death and reversed orprevented the loss of ChAT immunoreactivity in surviving motoneurons inthe ipsilateral nuclei. It also prevented any motoneuronal death in thecontralateral nuclei.

FIG. 22 shows Chat+ counts for facial motoneurons in 35 day old ratsafter a unilateral axotomy at 14 days of age. It shows the rescue of themotoneurons whose axons were transected (IPSI transection) and thecomplementary rescue of the small number of facial motoneurons that dieon the opposite side of the brainstem (Contra transection).

FIG. 23 sets out the data shown in FIG. 20 (leftmost two groups of bars)and includes data from some additional animals (group sizes increasedfrom 6 to 8 or more). It also shows that pargyline rescues themotoneurons (hatched bars, possibly more weakly than deprenyl as thegroups differ at the p<0.05 level). Further, a dose of 0.01 mg/kg ofdeprenyl was found to be just as effective as 10 mg/kg deprenyl inrescuing the motoneurons similar to the 0.01 mg/kg dose used with theMPTP model.

Animals lesioned at 14 days, treated for the next 21 days with 10 mg/kgdeprenyl (d14-35) and then left untreated until 65 days of age do notshow any further motoneuronal death (compare the third group of barsfrom the left to the corresponding bars in the second group that weresacrificed at 35 days of age when deprenyl treatment was still underway). This indicates that the rescue is permanent for the axotomizedmotoneuron i.e. the motoneurons do not begin to die when deprenyltreatment is discontinued after 21 days and there is not further deathover the next 30 days.

FIG. 23 also shows that rat motoneurons whose axons are transected at 1day of age have a greater amount of death than 14 day motoneurons andcannot be rescued by deprenyl. Therefore, it appears that some factormust reach maturity in the nervous system before deprenyl can beeffective and that factor appears between 1 and 14 days of age.

This is the first evidence that deprenyl can prevent the death ofmotoneurons and is consistent with the work indicating that deprenyl canreduce the death of axonally-damaged neurons. The death of axotomizedmotoneurons in immature rats is believed to reflect a dependency of themotoneurons for trophic support from the muscles they innervate (Crews,L. and Wigston, D. J.; J. Neurosci 10, 1643, 1990; Snider, W>D. andThanedar, S. supra). Recent studies have shown that some neuronotrophicfactors can reduce the loss of the motoneurons supporting that concept(Sendtner, M. et al, Nature 345,440, 1990). This study suggests thatdeprenyl has the capacity to activate some mechanism which compensates.for the loss of target derived trophic agents. Part of the action ofdeprenyl in neurodegenerative diseases may reflect a similarcompensation for reduced trophic support.

The finding of a small amount of motoneuronal death in the facialnucleus contralateral to a facial nerve transection is in accord withprevious reports of decreased numbers of axons in the intact nervecontralateral to the transection of a motor nerve (Tamaki K. Anat. Rec.56, 219, 1933) and a variety of other changes in contralateral nuclei(Pearson, C. A. et al. Brain Res. 463, 1988). Deprenyl completelyprevents the death of the contralateral motoneurons.

Axotomy initiates transient changes in protein synthesis in facialmotoneurons (Tetzlaff, W. et al. Neuro Sci. 8, 3191 (1988)) whichinclude a decrease in choline acetyl transferase (Hoeover, D. R. &Hancock, J. C. Neuroscience 15, 481, 1985). The small proportion ofsaline-treated motoneurons in the ipsilateral nuclei (16%) which lostChAT immunoreactivity probably reflects the surviving motoneurons thathad not recovered sufficient ChAT concentrations to be immunochemicallydetectable. Deprenyl prevented or reversed the loss of ChATimmunoreactivity in surviving motoneurons.

The dose of deprenyl (10 mg/kg) was sufficient to block the majority ofMAO-B activity and some MAO-A activity as well (Demarest, K. T.,Aazzaro, A. J. in Monoamine Oxidase: Structure, Function and AlteredFunctions (eds. Singer, T. P., Korff, R. W. and Murphy, D. L.) 423-340,Academic Press, New York, 1979) hence the reduction in motoneuron deathmay be due to MAO-B or MAO-A inhibition or may be independent of bothenzymes. However, it is expected that a 0.01 mg/kg deprenyl dose willproduce a reduction in motoneuron death similar to that obtained withthe 10 mg/kg dose. The 0.01 mg/kg dose does not produce any significantMAO-A or MAO-B inhibition indicating that the rescue with 0.01 mg/dgdeprenyl is not due to MAO-A or MAO-B inhibition.(See example 2) Thus,it is more likely that the reduction in motoneuron death will beindependent of MAO-B or MAO-A.

A recent study has shown that MAO-inhibitors may be more effective thendeprenyl in reducing the necrosis of dorsal striatal neurons after atransient interruption of the arterial blood supply to that region(Matsui, Y. and Kamagae, Y., Neurossci lett 126, 175-178, 1991). Yetdeprenyl doses (0.25 mg/kg) too low to produce inhibition of MAO-A butsufficient to product 20-75% inhibition of MAO-B in mice are aseffective as a 10 mg/kg dose in preventing the death of SNc neurons.MAO-B is largely concentrated in glial cells although present in someserotonergic and histaminergic neurons (Vincent, S. R. Neurosci 28,189-199 (1989); Pintari, J. E. et al. Brain Res. 276, 127-140, 1983).Since microglial cells show a proliferative response and astrogliarespond by an increase in protein synthesis to axotomy involving nearbymotoneurons, glial cells may be involved in deprenyl-induced preventionof neuronal death.

Example 5

Age Related Death of Mouse SNC neurons.

Studies were carried out to determine whether deprenyl preventsage-related death of mouse dSNc neurons using the procedures set out inTatton W. G. et al Neurobiol. Aging 1991; 12:5,543. The results areshown in FIG. 24.

As shown in FIG. 24, deprenyl does not prevent age-related death ofmouse DSN neurons.

Example 6

N-(2-aminoethyl)-4-chlorobenzamidehydrocloride having the followingformula ##STR1## was obtained from Research Biochemicals Incorporated,Natick MASS., U.S.A. (Cat. No. R-106, No. R016-6491) and was tested todetermine if it rescued immature axotomized motoneurons. A group offourteen day old rats received a unilateral facia nerve transection andwere treated with 10.5 mg/Kg N-(2-aminoethyl)-4-chlorobenzamide everyother day for 21 days. The rats were sacrified at 35 days of age andChAT+ immunochemistry was carried out as described in Example 4.

FIG. 25 contains the data shown in FIG. 23 and includes data from theanimals treated with N-(2-aminoethyl)-4-chlorobenzamide.

As shown in FIG. 25 the compound did not rescue the immature axotomizedmotoneurons (FIG. 25). It should be noted that the compound does nothave the triple bond C ending of the propargyl terminus of deprenyl andpargyline so that it attaches to a different part of the flavine portionof MAO-B. The propargyl attachment is permanent (irreversible inhibitionof MAO-B) while the N-(2-aminoethyl)-4-chlorobenzamide attachment isreversible and short lived.

Example 7

The (+) isomer and (-) isomer of deprenyl were tested to determinewhether the rescue of immature axotomized motoneurons wasstereospecific. A group of fourteen day old rats received a unilateralfacial nerve transection and were treated with 0.1 mg/Kg of the (-)isomer or (+) isomer of deprenyl every other day for 21 days. The ratswere sacrificed at 35 days of age and ChAT+ immunochemistry was carriedout as described in Example 4. As shown in FIG. 25, the (+) deprenyl ata dosage of 0.1 mg/Kg does not rescue the motoneurons. The rescueappears to be stereospecific to the (-) isomer. Thus, even through the(+) deprenyl has a terminal propargyl, the optical rotation relative tothe phenol ring and the intermediate portions of the molecule mayprevent attachment to the molecular site that initiates the rescue.

Example 8

Studies were carried out to determine the affect of deprenyl in ananimal stroke model. Rats were treated with carbonmonoxide and receivedglucose i.v. The carotid artery was then clamped and deprenyl wasadministered to the animals. The clamp was then removed causing strokein the animals. Deprenyl was also administered to a group of untreatedanimals one half hour after removal of the clamp. Positive neurons weredetermined in serial sections of the brain as described above. Deprenylwas found to reduce neuronal death and decreased the extent of damagedareas, in particular in the hippocampus.

We claim:
 1. A method for rescuing damaged nerve cells in a patient,comprising:administering to a patient having damaged nerve cells anamount of deprenyl, a pharmaceutically acceptable salt of deprenyl, oran ester of deprenyl such that rescuing of damaged nerve cells occurs inthe patient wherein the patient has damage resulting from a conditionselected from the group consisting of hypoxia, ischemia, stroke andtrauma.
 2. The method of claim 1 wherein deprenyl is administered to thepatient.
 3. The method of claim 2 wherein the deprenyl is (-) deprenyl.4. The method of claim 1 wherein the patient is human.
 5. A method forrescuing damaged nerve cells in a patient, comprising:topicallyadministering to a patient having damaged nerve cells an amount ofdeprenyl, a pharmaceutically acceptable salt of deprenyl, or an ester ofdeprenyl such that rescuing of damaged nerve cells occurs in the patientwherein the patient has damage resulting from a condition selected fromthe group consisting of hypoxia, ischemia, stroke and trauma.
 6. Themethod of claim 5 wherein deprenyl is administered to the patient. 7.The method of claim 6 wherein the deprenyl is (-) deprenyl.
 8. Themethod of claim 5 wherein the patient is human.
 9. A method for rescuingdamaged nerve cells in a patient, comprising:administering to a patienthaving damaged nerve cells an amount of deprenyl, a pharmaceuticallyacceptable salt of deprenyl, or an ester of deprenyl such that rescuingof damaged nerve cells occurs in the patient wherein the patient hasdamage resulting from a condition selected from the group consisting ofhypoxia, ischemia, stroke and trauma, wherein the deprenyl, apharmaceutically acceptable salt of deprenyl, or an ester of deprenyl isthe only active ingredient administered.
 10. The method of claim 9wherein deprenyl is administered to the patient.
 11. The method of claim10 wherein the deprenyl is (-) deprenyl.
 12. The method of claim 9wherein the patient is human.
 13. The method of claim 1 wherein thepatient is a non-human animal.
 14. The method of claim 5 wherein thepatient is a non-human animal.
 15. The method of claim 9 wherein thepatient is a non-human animal.